Further refinement of active specific immunotherapy (ASI) will require the precise identification of melanoma-associated antigens and epitopes recognized human T cells. T cells from immunized patients can serve as important reagents in such studies. Twenty-three novel melanoma genes have been identified by molecular subtraction between a melanoma and a squamous lung carcinoma. One of the genes whose mRNA was relatively restricted to melanomas has been studied: "gene 50". A 17 amino acid peptide of gene 50 was synthesized. It caused the proliferation of 4 CD4 clones from TIL of an immunized patient and was recognized by Th cells in 4 of 5 patients after ASI. The complete DNA sequence of gene 50 will be determined. It, and its various DNA domains, will be inserted into autologous lymphoblastoid cell lines with appropriate viral vectors, to test for epitopes causing proliferation of T-helper CD4 cells (Th epitopes) and those sensitizing for cytotoxicity by T-cytotoxic CD8 cells (Tc epitopes). Synthesis of a series of nonamers within a limited immunogenic domain may permit exact discrimination of melanoma epitopes. Transfection of complete gene 50 will be attempted with a retroviral vector, to obtain permanent target cells and specific stimulators of Tc and Th in vitro. Tc clones specific for the epitope will then be obtained from TIL or PBL. Their T cell receptors will be sequenced by PCR analysis with specific primers, to see whether what degree of correspondence exists between them and the epitope, particularly for Tc. The relevance of the immunogen encoded by gene 50 will be judged by determining how commonly patients given ASI develop Tc recognizing the gene 50 epitopes. The frequency of Tc before and after ASI will be measured by limited dilutions in patients of HLA-A2 and -non-A2 genotype. These studies may also indicate whether certain Tc epitopes can be recognized only in a particular MHC context. "Immortalization" of Tc clones recognizing specific epitopes, by means of transfected protein kinase C, may be helpful in exploring their properties. Once efficient screening and testing have been perfected, we will use recombinant vaccinia virus and later retroviral vectors into LCL to study the immunogenicity of 2 to 3 other novel melanoma genes that are relatively restricted to melanomas. This approach may elucidate human T cell-melanoma epitope interactions and in the process may lead to the development of a predetermined synthetic therapeutic melanoma vaccine.